Application of MdGATA15 gene in regulating and controlling synthesis of apple anthocyanin at high temperature

Abstract

The invention provides application of an MdGATA15 gene in regulating and controlling synthesis of apple anthocyanin at high temperature, and belongs to the technical field of biology. The expression level of the MdGATA15 gene in apples under high-temperature treatment is detected, the nucleotide sequence of the MdGATA15 gene is shown as SEQ ID NO: 1, it is found that the expression quantity of the MdGATA15 gene under high-temperature treatment can be induced to be remarkably increased, and then the content of anthocyanin in the apples is reduced, so that synthesis of the anthocyanin is increased by knocking out the MdGATA15 gene, the quality of the apples is improved, and the yield of the apples is increased. The problem that the apple color quality becomes poor at high temperature is solved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an application of an MdGATA15 gene in regulating the synthesis of anthocyanins in apple under high temperature. Background Art

[0002] Apple, as an important economic fruit tree, is widely planted around the world. China, as a major apple producing country, accounts for about 50% of the world's cultivated area and output.

[0003] Anthocyanins, as an important secondary metabolite, are widely involved in various life activities of plants, including defense or attraction of insects, resistance to invasion of pathogenic microorganisms, promotion of seed dispersal and plant reproduction. Anthocyanins make the flowers and fruits of plants present rich colors, and color is an important sensory quality of apples, affecting consumer preferences and market competitiveness of commodities.

[0004] In recent years, with global warming, high temperatures have occurred frequently and their impact has become increasingly serious. In the apple production process, rising temperatures will seriously inhibit the coloring process of apples. Therefore, understanding the molecular basis of the response of apple coloring molecular mechanism to high temperature is the key to solving the problem of apple color quality decline caused by global warming, and it will help us develop high temperature resistant varieties, optimize agricultural management measures, and formulate strategies to cope with global warming to ensure the stability of apple color quality. Summary of the invention

[0005] The purpose of the present invention is to improve the quality of apples and solve the problem of apple quality deterioration under high temperature by exploring the molecular basis of apple coloring and regulating the synthesis of anthocyanins.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides an application of an MdGATA15 gene in regulating the synthesis of anthocyanins in apple. The nucleotide sequence of the MdGATA15 is shown in SEQ ID NO: 1.

[0008] Preferably, the amino acid sequence of the protein encoded by the MdGATA15 gene is shown in SEQ ID NO: 2.

[0009] Preferably, the method for regulating the synthesis of anthocyanins in apple by the MdGATA15 gene is to increase the synthesis amount of anthocyanins by knocking out the MdGATA15 gene, and to reduce the synthesis amount of anthocyanins by overexpressing the MdGATA15 gene.

[0010] The present invention also provides an application of the MdGATA15 gene in regulating the synthesis of anthocyanins in apple under high temperature.

[0011] Preferably, the high temperature is 30°C to 40°C.

[0012] The present invention uses the cDNA obtained by reverse transcription of total RNA extracted from the callus of 'Wanglin' ​​apple as a template for PCR amplification. A new GATA transcription factor related to anthocyanin synthesis under high temperature in apple was cloned. Through Blast comparison analysis with related transcription factors in Arabidopsis, it was found that MdGATA15 was evolutionarily conservative and belonged to the B-type GATA factor. MdGATA15 was connected to an expression vector containing the strong promoter of CaMV35S using enzyme cutting and ligation technology. The MdGATA15 gene was overexpressed in callus tissue, and it was found that the anthocyanin content in the callus tissue overexpressing the MdGATA15 gene was reduced, and the expression level of anthocyanin synthesis-related genes was reduced. It was found that the expression level of MdGATA15 was significantly induced by high temperature in 'Otome' apples treated with high temperature. It shows that MdGATA15 can inhibit the accumulation of anthocyanins in response to high temperature signals. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the nucleic acid electrophoresis diagram of the MdGATA15 gene clone in Example 1;

[0014] Figure 2 The amino acid evolution tree analysis diagram of MdGATA15 in Example 1;

[0015] Figure 3 This is the subcellular localization map of MdGATA15 in Example 2;

[0016] Figure 4 A is the phenotype diagram of 'Otome' apple under the treatment of normal temperature 25°C and high temperature 35°C in Example 3; B is the statistical diagram of anthocyanin content and expression level of MdGATA15 gene under the treatment of normal temperature 25°C and high temperature 35°C in Example 3;

[0017] Figure 5 This is the phenotype diagram of the transgenic callus of MdGATA15 in Example 4;

[0018] Figure 6 This is the identification diagram of MdGATA15 transgenic callus in Example 4;

[0019] Figure 7 The left figure in the middle is a graph showing the anthocyanin content in the MdGATA15 transgenic callus in Example 4; the right figure is a graph showing the expression level of the MdGATA15 gene in the MdGATA15 transgenic callus (red is 'purple 3', WL is 'Wang Lin');

[0020] Figure 8This is the expression level diagram of anthocyanin synthesis-related genes in the MdGATA15 transgenic callus in Example 4 (red is 'Zi 3', WL is 'Wang Lin'). DETAILED DESCRIPTION

[0021] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0022] Example 1-MdGATA15 gene cloning and encoding amino acid phylogenetic tree analysis

[0023] 1. Cloning method of MdGATA15 gene

[0024] (1) Total RNA was extracted from the callus of 'Wanglin' ​​apple using a polysaccharide and polyphenol RNA extraction kit. The callus induction medium was: MS medium + 1.5 mg / L 2,4-D + 0.4 mg / L 6-BA + 20 g / L sucrose + 6 g / L agar powder. The pH of the induction medium was 5.8. 50 mg of 'Wanglin' ​​apple callus in good growth condition about 15 days after subculture was taken, ground into powder in liquid nitrogen, transferred to a 1.5 ml centrifuge tube, and 500 μL of lysis buffer SL (with 25 μL β-mercaptoethanol) was added. After deproteinization, DNA removal and rinsing, the callus was finally dissolved in 30 μL ddH2O.

[0025] The integrity of RNA was detected using 1% agarose, and the concentration and purity of RNA were determined using Nanodrop2000. The results showed that the RNA produced clear 28s and 18s bands in the electrophoresis diagram, the purity was 2.0 at A260 / A280 and 2.1 at A260 / A230, and the concentration was 1000 ng / ul.

[0026] (2) PCR amplification of the full length of MdGATA15

[0027] Take 5 μl of the above total RNA and perform reverse transcription according to the instructions of TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (Trans), and dilute the reaction solution 5 times as a PCR template.

[0028] Primers used for PCR cloning of MdGATA15

[0029] SEQ ID NO: 3F: ATGTTGGATCCAATCGATAAAGG;

[0030] SEQ ID NO: 4R: AGCATAGACAGAGCCATATGAGAGG;

[0031] PCR reaction system: total volume 25 μL, including 1 μL cDNA template, 2 μL dNTP, 1 μL each cloning primer, 5 μL 5×HFBuffer, 4.5 μL ddH20, and 0.5 μL Phusion high-fidelity enzyme.

[0032] The reaction procedure is: ①98℃ 3 min ②98℃ 30 s ③58℃ 30 s ④72℃ 1 min ⑤Repeat ②③④35 cycles ⑥72℃ 5 min ⑦Save at 4℃ The PCR product was purified using a gel recovery kit to obtain the target fragment, which was then stored at -20°C. The target fragment size was approximately 408 bp (e.g. Figure 1 The sequence is shown below).

[0038] The nucleotide sequence of the MdGATA15 gene is SEQ ID NO: 1:

[0039] ATGTTGGATCCAATCGATAAAGGATCGGAGTCAGAGGAAATGACTATCAAAACCCCAGATGCCGGTTCCTCGGAGGAAGGCTTTAAGAAAACCTGCGCCGACTGTGGAACCTCCAAAACCCCTCTGTGGAGAGGCGGTCCGGCGGGCCCAAAGTCTCTCTGTAATGCGTGCGGGATCAGAAGCAGGAAGAAGAGGAGGGCTATTTT GGGATTAAACAAAGAAAATCCCAACGATAAAAGGGGCAAGAAGAACAAGCAGCTCGGCGACGGCTTGAAGCAGCGGCTGTTGGCTTTGGGAAGAGAGGTTTTGATGCAGCGATCGACGGTGGAGAGGCAGCAGAGGAAGCTCGGCGAGGAAGAACAAGCGGCGGTGCTGCTGATGGCCCTCTCATATGGCTCTGTCTATGCTTAG;

[0040] The amino acid sequence of the protein encoded by the MdGATA15 gene is SEQ ID NO: 2:

[0041] MLDPIDKGSESEEMTIKTPDAGSSEEGFKKTCADCGTSKTPLWRGGP AGPKSLCNACGIRSRKKRRAILGLNKENPNDKRGKKNKQLGDGLKQRLL ALGREVLMQRSTVERQQRKLGEEEQAAVLLMALSYGSVYA.

[0042] (3) Preparation of carrier

[0043] The vector was prepared using a double enzyme digestion reaction system. The double enzyme digestion reaction system (20 μL) had the following component volumes: plasmid solution (pRI101) 10 μL, ddH2O 7 μL, 10×FastDigestBuffer 2 μL, Sal I 0.5 μL, Sma I 0.5 μL; plasmid solution (PHB) 10 μL, ddH2O 7 μL, 10×FastDigest Buffer 2 μL, Hind III 0.5 μL, BamH I 0.5 μL. Reaction conditions: 37°C metal bath, 15 min, to prepare the vectors pRI101 and PHB.

[0044] (4) Connection vector (homologous recombination connection)

[0045] The ligation was performed according to the full-form gold homologous recombination kit. The reaction system (10 μL) was as follows: 3 μL of the target fragment prepared in step (2), 5 μL of 2× Basic Assembly Mix, 1.5 μL of vector (pRI101 and PHB), and 0.5 μL of ddH2O. The components were mixed and reacted at 50°C for 15 min. After the reaction, the ligation product was obtained and placed on ice for the next transformation.

[0046] (5) Transformation of competent E. coli (DH5α)

[0047] Take out the DH5α competent cell from -80℃, put it on ice to melt, pipette the DH5α competent cell (35μL) and add it to the above-mentioned ligation product, flick to mix, and place on ice for 30min. Heat shock in a 42℃ metal bath for 90s, and quickly transfer to ice to cool for 2min. Add LB medium (300μL, without antibiotics) to the clean bench, and shake at 37℃ and 180rpm for 50min. Centrifuge at 10000rpm for 1min (carry out subsequent operations on the clean bench), discard the supernatant, retain about 100μL of the medium, and resuspend the bacteria. Pipette and mix, apply the bacterial solution to the LB medium (containing kanamycin (50mg / L)) plate, incubate at 37℃ overnight, and invert for 10h.

[0048] (6) Observe the size of the colony. After a single colony grows, pick a single colony and place it in LB liquid culture medium (500 μL, containing kanamycin (50 mg / L)) for shaking culture (37°C 180 rpm). After culturing for 6 hours until the bacterial solution becomes turbid, use it as a template for bacterial solution PCR verification, and use agarose gel electrophoresis to detect the PCR product. Select the better positive bacterial solution and send it to Qingdao Qingke Zixi Biotechnology Co., Ltd. for sequencing. The sequencing results are correct.

[0049] (7) Plasmid extraction: Use the Easy Pure Plasmid Miniprep Kit to extract plasmids from the correctly sequenced bacteria.

[0050] 2. MdGATA15 Phylogenetic tree analysis by MdGATA15 The amino acid sequence of Arabidopsis thaliana was used as a reference, and the phylogenetic tree was analyzed using MEGA 7.0 software. The amino acid sequence of GATA protein-related transcription factors in NCBI homepage Blast was found. MdGATA15 Evolutionarily conserved, belonging to the class B GATA transcription factor family (e.g. Figure 2 as shown).

[0052] Note: Transcription factors related to GATA proteins in Arabidopsis: AtBME3 (AT3G54800), AtGATA1 (AT3G24050), AtGATA10 (AT1G08000), AtGATA11 (AT1G08010), AtGATA12 (AT5G25830), AtGATA13 (AT2G28340), AtGATA14 (AT3G45170), AtGATA15 ( AT3G06740), AtGATA16 (AT5G49300), AtGATA17 (AT3G16870), AtGATA17L (AT4G16141), AtGATA2 (AT2 G45050), AtGATA23 (AT5G26930), AtGATA26 (AT4G17570), AtGATA27 (AT5G47140), AtGATA29 (AT3G20 750), AtGATA3 (AT4G34680), AtGATA4 (AT3G60530), AtGATA5 (AT5G66320), AtGATA6 (AT3G51080), A tGATA7(AT4G36240), AtGATA9(AT4G32890), AtGNC(AT5G56860), AtGNL(AT4G26150), AtHAN(AT3G50 870), AtHANL1 (AT2G18380), AtHANL2 (AT4G36620), AtZIM (AT4G24470), AtZIML1 (AT3G21175), AtZI ML2(AT1G51600), MdGATA15(LOC103403690), MdGATA9(LOC103454849), MdGATA11(LOC126615952).

[0053] Example 2 - MdGATA15 subcellular localization analysis method

[0054] 1. Obtaining the subcellular localization vector of MdGATA15

[0055] MdGATA15 was inserted into the PHB-GFP vector, and the endonucleases used were Hind III and BamH I. The connected MdGATA15-PHB-GFP vector was transformed into the competent Escherichia coli DH5α to obtain the recombinant plasmid, and cultured overnight at 37°C in a solid medium containing kanamycin. A single colony was picked and sequenced to identify the correctness of the recombinant plasmid. Finally, the positive recombinant plasmid with the correct identification was transformed into Agrobacterium tumefaciens GV3101-P19, and resistance screening was performed on YEP medium supplemented with 25 mg / L rifampicin and 50 mg / L kanamycin, and the strain was identified by PCR. The Agrobacterium containing the 35S::MdGATA15-GFP expression vector and the empty vector was finally stored in a -80°C refrigerator for use.

[0056] 2. Transient infection of tobacco leaves by Agrobacterium and localization observation

[0057] The tobacco used for positioning grew to 4-5 leaves. The night before, take 50 μL of the above Agrobacterium bacterial solution in 2 mL of YEP liquid medium containing resistance, and culture and activate Agrobacterium at 30°C overnight with shaking. The next day, take 1 mL of the activated bacterial solution and continue to culture in 30 mL of new YEP liquid medium containing resistance until OD 600 The value was 0.8, the bacterial solution was transferred to a 50mL centrifuge tube, centrifuged at 5000rpm for 10min, the supernatant was discarded, the bacterial solution was suspended with 30mL ddH2O, centrifuged again and the supernatant was discarded, and the OD value was adjusted to 0.8 with 10mL MES infection solution (500μMES (0.2M), 100μLMgCl2·6H2O (1M), 15μL acetosyringone (100mM), ddH2O to 10mL, pH 5.5), and the tobacco was injected after standing in the dark for 2h. The injected tobacco was cultured in the dark for 24h and then transferred to sunlight, and cultured for another 48h at 25℃. The fluorescence results were observed using an ultra-high resolution laser confocal microscope (Zeiss LSM880). Using the empty load as a control, MdGATA15 was localized in the cell nucleus (such as Figure 2 shown).

[0058] Example 3 High temperature treatment MdGATA15 Gene expression levels in 'Otome' apple 'Otome' apples were picked 120 days after flowering, and 30 fruits were placed in a light incubator at 25℃ and 35℃ with a light intensity of 20,000 lx for 9 days, and then photographed (e.g. Figure 4 The peels were harvested and total RNA of 'Otome' peel was extracted and reverse transcribed into cDNA. Real-time fluorescence quantitative PCR was performed using cDNA as a template to detect MdGATA15 Gene expression levels.

[0060] Real-time fluorescence quantitative PCR was performed using an iCycler iQ5 real-time quantitative PCR instrument (Bio-Rad, USA). The reaction system for real-time fluorescence quantitative PCR was 20 μL, containing 10 μL Maxima SYBR Green / Fluorescein QpcrMasterMix (Fementas), 1 μL cDNA template (reverse transcription product diluted 4 times), 7 μL ddH2O, and 1 μL of upstream and downstream primers (primer concentration 5 μmol / L). The reaction program for real-time fluorescence quantitative PCR was: 95°C pre-denaturation for 3 min, cycle parameters were 95°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 30 s, 40 cycles, and 72°C extension for 5 min. Apple Actin (AB638619.1) was used as the internal reference. 2 -ΔΔCT Quantitative data were analyzed by the PCR method, and all results were the average of three independent experiments.

[0061] The primers used to identify the MdGATA15 gene are as follows:

[0062] SEQ ID NO: 5F: AAGGATCGGAGTCAGAGGAA;

[0063] SEQ ID NO:6R: CCGCACGCATTACAGAGA.

[0064] The primers used to identify the MdActin gene are as follows:

[0065] SEQ ID NO:7F: TGACCGAATGAGCAAGGAAATTACT;

[0066] SEQ ID NO:8R: TACTCAGCTTTTGGCAATCCACATC.

[0067] The results showed that the expression level of MdGATA15 gene in 'Otome' apples treated with high temperature was significantly higher than that in the control group treated with 25℃ (e.g. Figure 4 (as shown in the right picture of B).

[0068] 2. Determination of anthocyanin content

[0069] 0.5 g of the apple peel harvested above was ground into powder in liquid nitrogen, and 5 mL of a mixed solution of 1% HCl: methanol solution (1:99 (volume ratio)) was added for extraction. The absorbance at 510 nm and 700 nm was measured (Shimadzu UV-2450, Kyoto, Japan). Anthocyanin content = ΔA × 5 × 0.005 × 1000 × 449.2 / (26900 × 0.5); ΔA = (A 510 -A 700 )(pH 1.0)-(A 510 -A 700 )(pH 4.5). The results are as follows Figure 4 As shown in the left figure of B, the anthocyanin content in the 'Otome' apples treated with high temperature was significantly lower than that in the control group treated with 25℃.

[0070] Example 4

[0071] To further verify the function of the MdGATA15 gene in anthocyanin accumulation, the present invention further provides a transgenic experiment of MdGATA15 in Example 4. Specifically, MdGATA15 was overexpressed and CRISPR / Cas9 knocked out in the callus of 'Zi 3' red flesh apple and 'Wang Lin' apple, and the changes in the anthocyanin content of the transgenic callus were observed.

[0072] 1. Construction of MdGATA15 overexpression vector

[0073] MdGATA15 was connected to the PRI101 overexpression vector by homologous recombination. The cloning primers containing homology arms of MdGAT A15 were: SEQ ID NO:9F:TTGATACATATGCCCGT CGACATGTTGGATCCAATCGATAAAGG; SEQID NO:10R:TTTACCCA TGAATTCGGATCCAGCATAGACAGAGCCATATGAGAGG, 5' containing Sal I restriction site, 3' containing BamHI restriction site. The PRI101 vector was double-digested with Sal I / BamHI, and the cloned MdGATA15 sequence was connected to the overexpression vector pRI101 to obtain a recombinant plasmid, and the correctness of the recombinant plasmid was identified by sequencing. Finally, the constructed recombinant plasmid was introduced into Agrobacterium LB A4404, and resistance screening was performed on YEP medium supplemented with 25 mg / L rifampicin and 50 mg / L kanamycin, and resistant Agrobacterium was identified by PCR. Finally, the correct Agrobacterium culture solution containing the 35S::Md GATA15 expression vector was stored at -80°C for later use.

[0074] 2. Construction of MdGATA15 CRISPR / Cas9 knockout

[0075] Log in to http: / / crispr.hzau.edu.cn / CRISPR2 / , search for high-scoring targets for MdGATA15 gene knockout, design corresponding primers, and use PCR amplification technology to amplify in PCBC-DT1 and PCBC-DT2 vectors. Connect the sgRNA containing the target to the final pHSE401 vector, transform Agrobacterium truncatum LBA4404 to obtain positive clone bacterial solution, and store the positive clone bacterial solution in a -80℃ refrigerator for infection.

[0076] The selected target 1 is SEQ ID NO: 11CCCTCTGTGGAGAGGCGGTC;

[0077] Target 2 is SEQ ID NO: 12CTCCGAGGAACCGGCATCTG;

[0078] The primers used were:

[0079] SEQ ID NO: 13MdGATA15-DT1-BsF: ATATATGGTCTCGATTGCCTCTGTGGAGAGGCGGTCGTT;

[0080] SEQ ID NO: 14MdGATA15-DT1-F0: TGCCTCTGTGGAGAGGCGGTCGTTTTAGAGCTAGAAATAGC;

[0081] SEQ ID NO:15MdGATA15-DT2-BsR:ATTATTGGTCTCGAAACTCCGAGGAACCGGCATCTGCAA;

[0082] SEQ ID NO:16MdGATA15-DT2-R0:AACTCCGAGGAACCGGCATCTGCAATCTCTTAGTCGACTCTAC;

[0083] 3. Transformation of apple callus using Agrobacterium tumefaciens:

[0084] The callus tissue of the 'Zi 3' red flesh apple and the callus tissue of the 'Wang Lin' apple were used. The callus tissue of the 'Zi 3' red flesh apple was cultured in a constant temperature incubator at 25°C under light conditions (intensity of 15000 lux), and the callus tissue of the 'Wang Lin' apple was cultured in a constant temperature incubator at 25°C under dark conditions. After the callus tissue was subcultured for 15 days, it was used for Agrobacterium transformation. The day before transformation, Agrobacterium was activated. 20 μL of Agrobacterium containing MdGATA15 overexpression and CRISPR / Cas9 vector was taken for activation (shaking overnight at 28°C), and 800 μL of the activated bacterial solution was added to 50 mL of YEP culture medium containing rifampicin (50 mg / L) and kanamycin (50 mg / L) and cultured to an OD value of 0.8. The cells were centrifuged at 5000 rpm for 8 min at room temperature, and the cells were collected and washed once with 30 mL of ddH2O. Finally, the OD value of the infection night (30 mL ddH2O + 30 μL 100 mM acetosyringone) was adjusted to 0.8, and 30 μL 100mM acetosyringone, immerse the callus in the impregnation solution, shake at 150rpm for 30min at room temperature, then dry the infected callus with sterile filter paper and culture it in MS medium, culture it in the dark at 25℃ for 48h, take out the callus and grow it in the selection medium containing 50mg / L kanamycin and 250mg / L carboxybenzene penicillin. The above steps are completed in a clean bench.

[0085] 4. Screening and identification of transgenic callus

[0086] PCR, RT-qPCR, western blotting and next-generation sequencing were used to identify the callus tissues grown in the screening medium. Three positive overexpression transgenic callus tissues (GATA15-OE-1 / 2 / 3) and three positive knockout transgenic callus tissues (GATA15-Cas9-2 / 3 / 6) were obtained. The obtained transgenic callus tissues were subcultured to observe the changes in the anthocyanin content of the transgenic callus tissues. The results are as follows: Figures 5 to 8 As shown, the anthocyanin content in the callus tissue overexpressing the MdGATA15 gene decreased, the expression level of the MdGATA15 gene increased, and the expression level of anthocyanin synthesis-related genes decreased; the anthocyanin content in the callus tissue knocked out the MdGATA15 gene increased, the expression level of the MdGATA15 gene decreased, and the expression level of anthocyanin synthesis-related genes increased. It can be seen that the expression of the MdGATA15 gene inhibits the expression of anthocyanin synthesis-related genes, thereby reducing the anthocyanin content.

[0087] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An application of the MdGATA15 gene in regulating the synthesis of anthocyanins in apple, characterized in that: The nucleotide sequence of MdGATA15 is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that The amino acid sequence of the protein encoded by the MdGATA15 gene is shown in SEQ ID NO:

2.

3. The use according to claim 2, characterized in that The method for regulating the synthesis of anthocyanins in apple by the MdGATA15 gene is to increase the synthesis amount of anthocyanins by knocking out the MdGATA15 gene, and to reduce the synthesis amount of anthocyanins by overexpressing the MdGATA15 gene.

4. Application of the MdGATA15 gene in regulating the synthesis of anthocyanins in apple under high temperature.

5. The use according to claim 4, characterized in that The high temperature is 30°C to 40°C.

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